Method of reducing cellular immune response involving T-cells using CD8-bearing antigen presenting cells

ABSTRACT

Specific and nonspecific immunomodulation, enhancement of cellular engraftment, and modulation of nonimmune cells are achieved by using various membrane-binding and soluble CD8 compositions.

This is a continuation of copending application Ser. No. 07/429,401filed on Oct. 31, 1989, abandoned.

This is a continuation-in-part of copending application U.S. Ser. No.07/323,770 (abandoned) filed Mar. 15, 1989.

FIELD OF THE INVENTION

The present invention relates to immunosuppression andimmunotolerization for the treatment of subjects in need of theabrogation of untoward immunological reactivities and of subjects inneed of the enhancement of cell, tissue and organ transplant survival.More particularly, it relates to the use of CD8 (hereinafter defined)and its derivatives as immunomodulators to effect said therapeuticobjectives. The present invention also relates to broader therapeuticuses for CD8's inhibitory ligand activity in the modulation of cellsoutside of the immune system.

BACKGROUND

In that application, included herein in its entirety by reference,antigen-specific (hereinafter referred to as "specific")immunosuppression and immunotolerization in subjects in need of theselective suppression of immune responses to defined antigens isachieved through the pharmaceutical use of CD8, and derivatives thereof.This therapeutic strategy is predicated upon our discovery that the CD8molecule inhibits immune and other cells that are being costimulatedwith certain secondary molecules (hereinafter referred to as "ligands";vide infra). Although a variety of immunosuppressive capabilities havebeen previously ascribed to T lymphocytes with a CD8-positive phenotype,the role of the CD8 molecule itself as a critical molecular determinantof inhibitory activity exerted by these cells was unknown until saiddisclosure. Moreover, prior to said disclosure of CD8's inhibitoryligand function, the only known function for CD8 was its molecularaccessory function, wherein it plays an obligatory role in T cellactivation through the T cell receptor complex.

At the present time, specific immunotolerization therapies, primarilycentered around the administration of specific antigen in associationwith additional substances, are relatively ineffective. Therapies fortransplant, allergic and other subjects in need of immunosuppressionmost commonly employ generalized, nonspecific immunosuppressive agents.These therapeutic agents, which include X-irradiation, cytotoxic drugs,cyclosporin A, corticosteroids, and antilymphocytic serum, suffer fromsignificant side effects involving multiple immune and nonimmune organs.Furthermore, in the case of clinical transplantation, no effectivestrategies for biochemically altering grafts in vitro to prolong theirsurvival in a host have been described.

An object of the present invention is to provide an effective processfor specific immunosuppression and immunotolerance induction, whichprocess comprises the use of CD8 compositions.

Another object of the present invention is to provide a process, usingCD8 compositions, for generalized, nonspecific immunosuppression, whichprocess suffers from fewer side effects than currently availableprocesses, and permits more specific targeting of organs of the immunesystem than current therapies.

Yet another object of the present invention is to provide a process forbiochemically altering grafts prior to transplantation, in a way whichenables them to evade immunological rejection mechanisms, and therebypromote their engraftment, which process comprises the use of CD8compositions.

Still another object of the present invention is to provide a processfor prevention of graft versus host disease following bone marrowtransplantation, which process comprises the use of CD8 compositions.

Still another object of the present invention is to provide a processfor selective modulation of nonimmune cells, which process comprises theuse of CD8 compositions.

Other objectives, features and advantages of the invention will be foundthroughout the following description and claims.

SUMMARY OF THE INVENTION

According to the present invention, there are provided compositionscomprising membrane-binding and soluble CD8 peptides, including thosegenetically engineered, and methods of use for immunomodulation andmodulation of nonimmune cells in vivo and in vitro. A pharmacologicallyactive CD8 composition comprises a CD8 peptide associated with one ormore secondary ligands that serve to direct CD8's inhibitory ligandactivity to specific target cells. This association between CD8 andsecondary ligands can be noncovalent and ensue simply from theirpresence on a common biomembrane (of a cell, liposome, planar membrane,pseudocyte, etc.), or covalent, through linkage in a CD8:ligandconjugate as part of a linear or branched polypeptide chimera. A broadarray of CD8:ligand combinations can be used, each of which permits thetargeting of CD8's modulatory activity to a specific subset of cells. Apreferred embodiment of the present invention, particularly applicablefor the purpose of specific T cell immunosuppression andimmunotolerization, comprises a CD8 composition in which CD8, or afunctional CD8 domain, is associated with a peptide derivative of amajor histocompatibility complex (MHC) protein. A defined nominalantigen peptide (NAP) can be secondarily associated with the MHCcomponent of said composition to permit the induction of specificimmunosuppression and immunotolerance for the parental proteinencompassing said NAP sequence. Another preferred embodiment of thepresent invention, particularly applicable for the purpose of mast celland basophil suppression in the treatment of immunoglobulin E(IgE)-related allergic disorders, comprises a soluble CD8:Fcε conjugatewherein CD8, or a functional CD8 domain, is associated with an IgE Fcdomain. Yet another preferred embodiment of the present invention,particularly applicable for the purpose of generalized, nonspecificimmunosuppression, comprises a soluble CD8:Fc conjugate, wherein CD8, ora functional CD8 domain, is covalently linked to an immunoglobulin(non-IgE) Fc domain. This CD8:Fc conjugate can be used to coat Fcreceptor (FcR)-bearing antigen presenting cells, and these cells, inturn, can be used to inhibit immune cells in a nonspecific fashion.Still another preferred embodiment of the present invention,particularly applicable for the purpose of prolongation of graftsurvival in a transplant recipient, comprises a process wherein amembrane-binding CD8 peptide is used to coat graft cells prior totransplantation, to promote engraftment in a transplant recipient. Stillanother preferred embodiment of the present invention, particularlyapplicable for the purpose of prevention of graft versus host diseasefollowing bone marrow transplantation, comprises a process wherein atherapeutic biomembrane preparation comprising a CD8 peptide and a MHCpeptide corresponding to the transplant recipient's haplotype, or aCD8:MHC conjugate, is used to treat bone marrow cells in vitro prior totransplantation, to inhibit alloreactive immune cells in the donor cellpopulation.

THE PREFERRED EMBODIMENTS

The present invention is directed to methods for cellular modulation,with a focus on cells of the immune system. The compositions and methodsfor specific immunosuppression, specific immunotolerization andgeneralized, nonspecific immunosuppression are applicable to, but notrestricted to, the clinical settings of transplantation and autoimmune,hypersensitivity, allergic and other immunological disorders.

The present invention is predicated upon our discovery, as disclosed byus in U.S. Ser. No. 07/323,770, that the CD8 molecule can function as aninhibitory ligand. The inventors of the present invention previouslydeveloped a methodology for stable gene transfer into nontransformed,cloned human T cells (Proc. Natl. Acad. Sci. U.S.A. 85:4010-4014, 1988).This methodology, in turn, enabled the first linking of antisensemutagenesis and T cell cloning technologies. When applied to CD8, inearlier studies, to create T cell clonal phenocopies of null mutationsfor CD8, this transfection technology permitted a definitivedemonstration of CD8's function as an obligatory accessory molecule forthe specific activation and killing mediated by CD8-positive cytotoxic Tcells (J. Exp. Med. 168:1237-1245, 1988). Another byproduct of ourtransfection technology, and specifically of our ability to produceCD8-negative antisense mutants, is the insight into CD8's previouslyunsuspected role in the suppression mediated by CD8-positive T cells, asdisclosed in the present invention.

Specifically, we have established that a native or geneticallyengineered CD8 peptide can inhibit T cells and other cells when said CD8peptide is associated with a second ligand that would otherwise functionas a cellular activator. For instance, in the case of T cells, thesecond ligand can be an allo-MHC molecule or a specific (processed)antigen associated with a self-MHC molecule. CD8's immunomodulatoryactivity has been assessed experimentally by our group using severaltypes of in vitro cellular proliferation and cytotoxicity assays,employing a variety of sense and antisense CD8 transfectants andcontrols. Cells for these studies were obtained from subjects JH (HLAhaplotype:A2,3;B7,44;DR2,4), DK (HLA haplotype A2,24;B13,50;DR2,7), andMW (HLA haplotype A1,31;B8;DR3). Our findings include the following:

(i) The proliferative response of responding cells to irradiated,allogeneic stimulator cells in mixed cell cultures is dependent upon theabsence of CD8 on the irradiated stimulators. In one such experiment,10⁵ responder peripheral blood mononuclear cells (PBMC) from DK werecombined with stimulators comprising either 2×10⁴ irradiated (5000 R),syngeneic cells of the cloned, CD8-positive T cell line JH.ARL.1(derived from JH and with known allospecificity for HLA-B35) or 2×10⁴irradiated (5000 R), CD8-negative antisense CD8 transfectant derivativesof the JH.ARL.1 line, in quadruplicate wells of a 96-well flat-bottommicrotiter plate in RPMI 1640 medium with 10% fetal bovine sera. Cellswere cocultured for 4-7 days at 37° C., adding 1 μCi of [³ H]-thymidineto each well during the last 18 h. The cells were harvested, and theradioactivity incorporated was counted. A proliferative response wasobserved for the CD8-negative, but not for the CD 8-positive, stimulatorat all time-points. This was confirmed using a sense, instead of anantisense, transfection approach. In one such experiment, 10⁵ responderPBMC from MW were combined with stimulators comprising either 2×10⁴irradiated (15,000 R), nontransfected K562 human eyrthroleukemia cellsor K562 cells transfected with an irrelevant expression construct, bothof which are CD8-negative, or 2×10⁴ irradiated (15,000 R) sense CD8 K562transfectants (CD8-positive) in the proliferation assay as described.Again, only the CD8-negative, but not CD8-positive, cells were able tostimulate proliferation.

(ii) The proliferative response of responding cells to irradiated,allogeneic stimulator cells and their capacity for cytotoxicity againstallogeneic target cells can be inhibited by the simultaneous addition ofthird party cells, if the latter cells bear both CD8 and a specificalloantigen that is recognized by the responding cells, and thus suchcells function in an immunosuppressive "veto-like" capacity. In one suchexperiment, 10⁵ responder PBMC from DK were combined with 5×10⁴irradiated (5000 R) stimulator PBMC from JH and varying numbers ofcloned T cells as putative inhibitors, originating from JH, comprisingeither CD8-positive or CD8-negative (antisense) JH.ARL.1 transfectants.Inhibition of the proliferative response was observed only for theCD8-positive third party cells, and the potency of the inhibition wasdemonstrated by the finding of 33% and 78% inhibition atinhibitor:stimulator cell ratios of 1:500 and 1:5, respectively. Absenceof inhibition upon combining JH responders, DK stimulators, and JHcloned (CD8-positive) T cell inhibitors established that a specificrecognition event is required between responders and CD8-positiveinhibitors in order for inhibition to occur. We further demonstratedCD8-dependent inhibition of cytotoxic T cell generation in MLR cultures.Allogeneic cultures were set up in 24-well plates in a volume of 2 ml ofRPMI 1640 containing fetal bovine sera. 10⁶ responder PBMC from MW,5×10⁵ irradiated (5000 R) stimulator PBMC from JH, and 10⁵ irradiated(CD8-positive or CD8-negative antisense phenocopies) cloned JH.ARL.1cells were added per well. After incubation for 6 days at 37° C., cellswere harvested, and dead cells removed by histopaque density gradientcentrifugation. A [⁵¹ Cr]-release assay (Cell. Immunol. 88:193-206,1984) was performed with EBV-transformed JH (LCL) B lymphocytes astargets. Inhibition was evident only with CD8-positive third partycells, and maximal inhibition was achieved when these cells were addedat the initiation of the cultures or by day 2. These findings withJH.ARL.1 lymphoid cells as inhibitors were confirmed with non-lymphoidK562 cells as stimulators and inhibitors. In one such experiment, 10⁵responder PBMC from JH, 5×10⁴ irradiated (20,000 R) K562 stimulators,and putative inhibitors, comprising either irradiated CD8-negative K562cells or CD8-positive sense K562 transfectants were used. MarkedCD8-dependent inhibition was observed with as few as 5×10² inhibitors.In another experiment, irradiated (15,000 R) immortalized human bonemarrow stromal cells were used in place of K562 cells, with analogousresults.

(iii) The proliferative response to responding cells to irradiated,allogeneic stimulator cells can be blocked by pretreatment of theresponding cells with irradiated, or otherwise metabolicallyinactivated, third party "tolerogenic" cells, if the latter cells bearboth CD8 and a specific alloantigen that is recognized by the respondingcells. In one such experiment, 10⁵ PBMC responders from JH wereincubated with fixed (air-dried, with or without post-treatment with 2%paraformaldehyde in phosphate-buffered saline for 2 h at 37° C.)CD8-positive or CD8-negative K562 or human bone marrow stromal celltransfectants for 48 h in RPMI 1640 supplemented with 10% fetal bovinesera, in quadruplicate 96-well plates. PBMC so pre-treated wererecovered, stimulated with CD8-negative counterparts for 18 h in thepresence of 0.5 μCi/well [³ H]-thymidine, and [³ H]-thymidineincorporation was measured. The data indicated a marked and specificCD8-dependent tolerization amidst the responders.

Hence, the inhibition directed by CD8 can be used to both immunosuppressand immunotolerize. Moreover, assays similar to the ones described haveindicated that CD8-mediated inhibition is applicable for theimmumomodulation of responses to specific antigens other thanalloantigens, as well as for the modulation of nonimmune cells (videinfra).

Further experiments have elucidated the functional requirements for theCD8 ligand itself. Two forms of CD8 are known to be present on human Tlymphocyte surfaces: an α:α homodimer and an α:β heterodimer. CD8α(herein referred to as "CD8") can function as an inhibitory ligand as amonomer, and hence the CD8β chain is not required for inhibition. CD8βrequires CD8β in order to be efficiently expressed on the cell surfacein native cellular settings. The CD8 peptide need not be expressed onimmune cells per se, and hence T cell-specific factors are not requiredfor CD8-mediated inhibition. Furthermore,glycoinositolphospholipid-modified CD8, when anchored to membranes, andeven CD8 anchored to fixed cells, both maintain the inhibitory capacityof the native CD8 molecule. Hence, CD8-mediated inhibition is dependentupon the physiological status of the responding (target) cell, but isindependent of the physiological status of the inhibitory cell. Thelatter point lays the groundwork for developing compositions comprisedof membrane-binding CD8 derivatives linked to liposomes or othermembranous therapeutic vehicles or of soluble CD8 derivatives.

One embodiment of a CD8 composition according to the present inventioncomprises the complete extracellular region of CD8 [encompassing aminoacids 1 (Ser) through 160 (Cys) of processed human CD8; for CD8 codingsequence, see Littman, D. R., et al., Cell 40: 237-246, 1985]. Analternative CD8 composition is comprised of a functional domain withinthe extracellular region of CD8, such as one corresponding to theimmunoglobulin V homologue region of CD8 [encompassing amino acids 1(Ser) through 114 (Ala) of processed human CD8]. Protein engineeringstrategies using recombinant DNA tools for molecularly dissecting aprotein such as CD8, to define functionally active subcomponents, arewell known to those familiar with the art, and hence can be used todefine additional functional CD8 peptide derivatives.

Another embodiment of a CD8 peptide composition according to the presentinvention comprises a CD8:ligand conjugate, wherein CD8, or a functionalpeptide derivative thereof, is covalently linked to one or moresecondary ligand molecules, the latter permitting the selectivetargeting of, and providing a costimulatory signal to, a specific subsetof cells. The second ligand molecule of such a "bipartite CD8 ligand"can be peptidic or nonpeptidic in nature. In the case of peptideligands, the CD8 and second ligand peptides can be linked in a linear orbranched polypeptide chimera (vide infra). Additional ligands (third,etc.) can be similarly linked, and by utilizing such a "multipartite CD8ligand," the effectiveness of the invention can be enhanced.Furthermore, membrane-binding or soluble forms can be produced. Severalexamples of CD8:ligand conjugates will now be cited, which serve toillustrate, but in no way restrict, the types of such CD8-basedconjugates that can be produced and used for cellular modulation.

One example of CD8:ligand conjugate is a CD8:MHC conjugate, wherein aCD8 peptide is covalently linked to a class I or class II MHC protein,or a functional peptide derivative thereof. Functional MHC peptidederivatives are comprised of those domains that are sufficient, andmaintain the capacity in the synthetic peptide, for constituting anominal antigen peptide (NAP) binding site. In the case of class I MHC,which is composed of a polymorphic, transmembrane α heavy chain and anoncovalently-associated, nonpolymorphic, non-membrane-anchored β₂-microglobulin light chain, the α₁ and α₂ extracellular domains of the αheavy chain together constitute a NAP binding site (Bjorkman, P. J., etal. Nature 329: 506-512, 1987). In the case of class II MHC, which iscomposed of noncovalently-associated, polymorphic, transmembrane α and βchains, the α₁ domain of the α chain and the β₁ domain of the β chainare sufficient for constituting a NAP binding site (Brown, J. H., et al.Nature 332: 845-850, 1988). NAPs can be associated, noncovalently orcovalently, with the CD8:MHC conjugate to confer antigenic specificityto said conjugate, and permits targeting of specific T cells.

A second example of a CD8:ligand conjugate is a CD8:unprocessed antigenconjugate, wherein a CD8 peptide is covalently linked to an unprocessedantigen, the latter constituting a ligand for immunoglobulins on thesurface of specific B cells. Examples of unprocessed antigens includeallergens, such as benzyl-penicilloyl, insulin, ovalbumin, lactalbumin,grass pollens, ragweed pollen, ragweed antigen E, tree pollens, beevenom, snake venom, and house dust mite, and self-antigens. Suchconjugates permit targeting of specific B cells and are used to induceantigenic unresponsiveness and tolerance in humoral immune responses.

A third example of a CD8:ligand conjugate is a CD8:Fc conjugate, whereina CD8 peptide is covalently linked to the Fc domain of an immunoglobulinmolecule, or a functional, Fc receptor (FcR)-binding derivative thereof.Fc domains corresponding to any of the immunoglobulin isotypes can beemployed for this purpose. Such a conjugate permits targeting ofspecific classes of Fc-receptor bearing cells. This, in turn, can serveone of two purposes from a functional standpoint. For certainFcR-binding cells, an inhibitory signal will be transduced by the CD8:Fcconjugate. Even in the absence of an inhibitory effect, the CD8:Fcconjugate will bind to the surface receptors, serving to coat the cellsurface with CD8. This, in turn, serves to convert an FcR-positiveantigen presenting cell into an inhibitory cell.

A fourth example of a CD8:ligand conjugate is a CD8:Fv conjugate,wherein a CD8 peptide is covalently linked to the Fv (antigen-binding)domain of an immunoglobulin molecule, or an Fv-containing peptide. TheFv component confers specificity for specific cell surface-associatedmolecules bound by the Fv component, and thereby permits targeting ofspecific cells.

A fifth example of a CD8:ligand conjugate is a CD8:cytokine conjugate,wherein a CD8 peptide is covalently linked to a peptidic cytokine. Abroad array of cytokines can be used for this purpose, including colonystimulating factors, interleukins and hormones. Such conjugates permittargeting of specific cytokine receptor-bearing cells.

A sixth example of a CD8:ligand conjugate is a CD8:lectin conjugate,wherein a CD8 peptide is covalently linked to a lectin. A broad array oflectins can be used for this purpose, including conconavalin A andphytohemagglutinin. Such conjugates permit targeting of specific normaland transformed cells bearing defined, lectin-reactive carbohydratespecificities on their surfaces.

A seventh example of a CD8:ligand conjugate is a CD8:anti-Id conjugate,wherein a CD8 peptide is covalently linked to an anti-idiotypic(anti-Id) mimic of a second ligand, such as one of those describedheretofore. Additionally, an anti-Id can be used as a mimic of CD8itself in any of the CD8 compositions described heretofore.

CD8 peptides, comprising either CD8 sequences only or CD8 sequencescoupled to peptide or nonpeptidic ligands in CD8:ligand conjugates, canbe soluble or membrane-binding. Coding sequences can be geneticallyengineered to create soluble forms by introducing a translational stopcodon into the coding sequences of CD8 and peptide ligands, upstream ofthe hydrophobic transmembrane domains, using site-specific mutagenesistechnologies. Coding sequences can be genetically engineered to createmembrane-binding forms by linking, or retaining the linkage of, thecoding sequences of CD8 and secondary peptide ligands to: 1) codingsequences for hydrophobic extension peptides of transmembrane proteins;or 2) coding sequences that direct glycoinositolphospholipidmodification of peptides inside cells. Aglycoinositolphospholipid-modified CD8 peptide represents a preferredembodiment of a membrane-binding CD8 peptide, according to the presentinvention, since it can be readily incorporated into biomembranes whenexogenously added to them.

Linear polypeptide chimeras, in the forms ofglycoinositolphospholipid-modified protein intermediates and CD8:ligandconjugates, as disclosed in the present invention, can be readilyproduced by recombinant DNA technology. Chimeric transcriptionalcassettes can be assembled using restriction endonuclease site overlapor the polymerase chain reaction (PCR)-based splice-by-overlap-extension(Horton, R., et al., Gene 77: 61-68, 1989) methodologies. To produceglycoinositolphospholipid-modified peptides, the coding sequence for thepeptide of interest is linked in-frame to the coding sequence for the 3'end of a protein that naturally undergoes glycoinositolphospholipidmodification, such as decay accelerating factor (DAF). The chimericprotein produced in this way undergoes glycoinositolphospholipidmodification inside the cell. Thisglycoinositolphospholipid-modification process was discovered by one ofthe inventors of the present invention (M.L.T.), and it was firstapplied to CD8. (Tykocinski, M., et al., Proc. Natl. Acad. Sci. U.S.A.85: 3555-3559, 1988). To produce CD8:ligand conjugates, the codingsequences for a CD8 peptide, a suitable linker peptide, and the ligandpeptide are tandemly linked in-frame. Choice of promoters, for thechimeric gene transcriptional cassette, vectors and host cells willdictate the nature of post-translational modifications introduced intothe chimeric protein and the quantity of protein produced. For instance,baculovirus promoters and vectors can be used in insect host cells toproduce large quantities of glycosylated CD8 compositions.

We have produced various genetic constructions for generatingglycoinositolphospholipid-modified and soluble CD8 peptides. As startingmaterial for these genetic constructions, we either PCR-cloned specificmRNAs from reverse transcribed poly(A+)RNA or obtained cDNA clones ofhuman CD8 (for nucleotide sequence, see Cell 40: 237-246, 1985; ATCCdeposit no. 59565), human DAF (for nucleotide sequence, see Proc. Natl.Acad. Sci. USA 84: 2007-2011, 1987), human class I MHC α heavy chain ofthe A2 haplotype (for nucleotide sequence, see J. Immunol.134:2727-2733, 1985), human GM-CSF (Science 228: 810, 1985; Proc. Natl.Acad. Sci. USA 82: 4360, 1985; ATCC deposit nos. 39754, 57595 and59171), human IgG1 heavy chain γ1 (for nucleotide sequence, see NucleicAcids Res. 10: 4071-4079, 1982), a human IgE heavy chain ε (fornucleotide sequence, see Cell 29: 691-699, 1982). Examples of CD8peptides include, but are not restricted to, the following:

(i) Production of a glycoinositolphospholipid-modified CD8 peptide,encompassing the complete extracellular domain of CD8 (through asp 161),by a restriction endonuclease-based methodology. Specifically, the 3'end of DAF cDNA is cut at the Ava II site (nucleotide position 858),this site is blunted by filling-in with Klenow fragment, and the CD8coding segment cut at the EcoRV site (nucleotide position 609) isblunt-end ligated to the filled-in Ava II site of DAF. This creates aCD8:DAF chimera, which undergoes glycoinositolphospholipid-modificationinside cells (Tykocinski, M. L., et al. Proc. Natl. Acad. Sci. USA 85:3555-3559, 1988).

(ii) Production of a glycoinositolphospholipid-modified CD8 peptide,encompassing the immunoglobulin V-homologue domain of CD8 (through ala114), by a splice-by-overlap extension methodology. Specifically, theCD8 sequence (spanning nucleotide positions 31 through 470) isPCR-amplified (denaturing 94° C., 2'; annealing 50° C., 2'; polymerizing72° C., 2'; using Perkin Elmers-Cetus, Inc. thermal cycler and Gene-Ampkit) with the oligonucleotide primers a[5'-GGATCCAAGCTTCTCGAGAGCTTCGAGCCAAGCAGC-3'] andb[5'-GAACTGTTGGTGGGACCGCTGGCAGGAAG-3'], and the DAF sequence (spanningnucleotide positions 859 through 2008; starting at val 258) isPCR-amplified with the oligonucleotide primersc[5'-CAGCGGTCCCACCAACAGTTCAGAAACCT-3'] andd[5'-GAGCTCGAGAAGCTTTGGGATCATTTATTT-3']. Primer a adds BamHI, Hind III,and XhoI sites to the 5'-end, and primer b adds Hind III, XhoI, and SacI sites to the 3'-end. Primers b and c each bridge both CD8 and DAFsequence, and are complementary to each other at their 5' ends. Hence,the separate CD8 and DAF PCR products, when diluted (1:100), combined,denatured and reannealed, yield a subset of chimeric CD8:DAF molecules,which are then PCR-amplified with the a and d primers (94° C., 2'/37°C., 2'/72° C., 2' for 10 cycles; 94° C., 2'/50° C., 2' /72° C., 2' forthe next 20 cycles). The CD8:DAF chimera is gel-purified, digested withHind III at its ends and ligated into the Hind III site of theBluescript prokaryotic cloning vector (Stratagene, Inc., San Diego,Calif.). An alternative version of a chimeric CD8:DAF gene, in which thecoding sequences for the membrane-proximal O-glycosylation region of DAFare omitted, is produced by substituting primers b and c for primerse[5'-CACTTCCTTTATTTGGCGCTGGCAGGAAGACC-3'] andf[5'-CAGCGCCAAATAAAGGAAGTGGAACCACT-3'], respectively. The f and g primerpair PCR amplifies the DAF sequence spanning nucleotide positions 1018through 2008 (starting at pro 311).

(iii) Production of a soluble CD8 peptide, encompassing the V-homologuedomain of CD8 (through ala 114), by a PCR-based site-directedmutagenesis methodology (Ho, S.N., et al. Gene 77:51-59, 1989).Specifically, the CD8 sequence (spanning nucleotide positions 31 through470) is PCR-amplified with the oligonucleotide primers a (as above) andg[5'-GAGCTCGAGAAGCTTTTACGCTGGCAGGAAGACCGG-3']. The g primer inserts astop codon immediately downstream of ala 114 and adds Sac I, Xho I andHind III sites to the 3' end. The PCR-amplified DNA segment is digestedwith Hind III and ligated into the Bluescript cloning vector.

(iv) Production of a soluble CD8:MHC conjugate, encompassing thecomplete extracellular domains of both CD8 and the class I α heavy chainof the A2 haplotype, by a splice-by-overlap-extension methodology.Specifically, primers, with suitable complementary overlap sequences,are used to PCR-amplify and link in-frame the coding sequences for theα₁ -α₂ -α₃ extracellular multidomain unit of the A2 class I α heavychain (through trp 274), a linker peptide with minimal secondarystructure, and the extracellular domain of CD8 (through asp 161). Thelinker peptide is comprised of the repeating unit(Gly.Gly.Gly.Gly.Ser)₃, and it is generated from complementaryoligonucleotides produced on an oligonucleotide synthesizer (PCR-Mate,Applied Biosystems, Inc.). A genetically engineered class I β₂-microglobulin can be secondarily associated with the class I α chain.Alternatively, an α₁ -α₂ (through thr 182), instead of an α₁ -α₂ -α₃,MHC multidomain unit is incorporated into such a conjugate. Positioningof the α₁ -α₂ MHC multidomain unit at the amino terminus of either ofthese linear polypeptide chimeras permits the component α₁ and α₂ unitsto fold, as they do in their native state, to constitute anantigen-binding pocket.

(v) Production of a soluble CD8:GM-CSF conjugate, encompassing thecomplete extracellular region of CD8 (through asp 161) and completeGM-CSF (through glu 127), by a splice-by-overlap-extension methodology.An analogous approach to that used for the CD8:MHC conjugate is used,wherein the coding sequences for GM-CSF, a flexible linker peptide, andCD8's extracellular region are linked. The capacity for binding toGM-CSF receptors is retained by this chimera.

(vi) Production of a soluble CD8:Fcγ1 conjugate, encompassing thecomplete extracellular domain of human CD8 (through asp 161) and the Fcregion of the human IgG1 heavy chain (γ1), by asplice-by-overlap-extension methodology. This CD8:Fc conjugate differsfrom the CD8:MHC and CD8:GM-CSF conjugates, detailed as examples above,in two ways: 1) this CD8:ligand conjugate has the CD8 componentpositioned at the amino terminal end of the chimeric protein; and 2) theCD8 and ligand components of this CD8:ligand conjugate are connected toeach other without an intervening linker peptide. Primers, with suitablecomplementary overlap sequences, are used to PCR-amplify and linkin-frame the coding sequences for the complete extracellular domain ofCD8 and the complete constant region of the human γ1 heavy chain,encompassing the C_(H) 1.C_(H) 2.C_(H) 3 multidomain unit starting atala 114. Specifically, the CD8 sequence (spanning nucleotide positions31 through 611) is PCR-amplified with the primers a andh[5'-TGGTGGAGGCATCACAGGCGAAGTCCAG-3'], and the γ1 sequence with primersi[5'-CTGTGATGCCTCCACCAAGGGCCCATCGGT-3'] andj[5'-GTACGTGCCAAGCATCCTCGTGCGACCG-3']. This CD8:Fc conjugate can beisolated by staphylococcus protein A-sepharose chromatography, by virtueof the retained capacity of the Fc domain of the disulfide-linkedpolypeptide chimera dimer to bind to protein A. In a similar fashion, asoluble CD8:Fcε conjugate is assembled, incorporating the C_(H) 1.C_(H)2.C_(H) 3.C_(H) 4 multidomain unit of human IgE. Specifically, the CD8segment (spanning nucleotide positions 31 through 611; ending at asp161) is PCR-amplified with primers a and k[5'-GTGTGGAGGCATCACAGGCGAATCCAG-3'] and ε(spanning nucleotide positions98 through 1847; starting with ala 114) is PCR-amplified with primers1[5'-CTGTGATGCCTCCACACAGAGCCCATCCGTCTTC-3'] andm[5'-GTCATTGCAACAGTGGACAGAAGGTCT-3'].

Branched polypeptide chimeras, in the form of CD8:ligand conjugates, canbe readily produced by template-assembled synthetic peptide (TASP)technology (Mutter, M., Trends Biochem. Sci. 13:260-265, 1988). By thisprocess, the peptide units are synthesized separately and covalentlycoupled to a multifunctional carrier, such as a core peptide, usingchemical coupling reagents. For example, a cyclic decapeptide analogueof gramicidin S, in which two antiparallel β-sheet segments(lys-ala-lys) are linked by two β-turns, can be used as a core peptide.Segment condensation strategies can be used to attach CD8 and secondaryligand peptides to the ε-amino groups of the 4 lysine side chains.Alternatively, CD8 and ligand components can be covalently linkeddirectly to each other in branched structures using chemicalcross-linking reagents. By this methodology, for example, CD8 and Fcdimers can be directly linked. Branched, as opposed to linear,polypeptide chimeras are particularly well-suited for providing formultivalent CD8:ligand conjugates (vide infra), with varying CD8 toligand ratios.

To facilitate the biochemical isolation of the various CD8 compositionsdisclosed heretofore, the primary sequence of the CD8 peptide, or in thespecial case of CD8:ligand conjugates, the primary sequence of eitherthe CD8 or ligand peptides, can be altered through genetic engineeringstrategies. A particularly useful alteration is the insertion of two ormore neighboring histidine residues. This insertion can be in the aminoor carboxy terminus of the peptide. Additionally, for CD8:ligand linearpolypeptide chimeras, the histidines can also be inserted into thelinker peptide, and for CD8:ligand branched polypeptide chimeras, thehistidines can also be inserted into the core peptide. Histidine residueinsertions can be readily accomplished by the splice-by-overlapextension methodology, by incorporating histidine-encoding CAT and CACtriplet codons into the PCR primers at suitable locations in the codingsequence. Histidine-modified proteins can be efficiently andquantitatively isolated by nickel-sepharose chromatography. Thehistidine-nickel interaction is based upon protonation, and hence thisinteraction can be reversed, for purposes of peptide elution, through asimple pH shift. Other primary sequence modifications, such as theinsertion of reactive amino acids for specific chemical couplingreagents, can also be performed. Alternatively, more conventional, andconsiderably less efficient biochemical isolation strategies can beemployed, including those based upon immunoaffinity (e.g., anti-CD8primary antibodies).

Another embodiment of a CD8 composition according to the presentinvention comprises biomembranes coated with CD8 (and a second ligand)or CD8:ligand peptides. These biomembranes can be in the form of, butare not restricted to, cells, liposomes, planar membranes or pseudocytes(Goldstein, S. A., et al. J. Immunol. 137:3383-3392, 1986). Cells thatnaturally bear CD8, such as CD8-positive T lymphocytes, or that arecoated and/or genetically engineered to bear CD8 can be alternativelyutilized. A cellular form that is particularly well-suited, as atherapeutic agent, for modulating cells in the blood compartment areautologous, or heterologous blood group-matched, erythrocytes coatedwith CD8 peptides. The applicability of liposomes for pharmaceuticalpurposes has been documented extensively in U.S. patent filings.CD8-coated liposomes, as disclosed in the present invention, canadditionally be internally loaded with organic and inorganicconstituents, such as cytokines and toxins, to be targeted to specificcells. A glycoinositolphospholipid-modified CD8 peptide is a preferredmembrane-binding CD8 composition according to the present invention, tobe used for coating biomembranes, since said peptide, so modified, canbe readily incorporated into biomembranes in the presence of low,non-lytic concentrations of detergents. Both free cells and cellsembedded in a tissue matrix can be coated withglycoinositolphospholipid-modified CD8 peptides. Another coating processentails the use of cross-linking chemical reagents to bind a CD8 peptideto biomembranes. Various processes for covalently coupling peptides toliposomes have been disclosed (see, for example, U.S. Pat. Nos.4,565,696 and 4,762,915). Yet another means of producing cells coatedwith a CD8 peptide is through the use of gene transfection technology.By any of these coating processes, multiple additional molecules can beadded to the biomembrane to enhance the biological potency of the CD8peptide. The various acellular therapeutic biomembrane preparationsdescribed heretofore can be stored in a dehydrated form, and packagedinto kits, for pharmaceutical use.

One embodiment of a CD8-mediated therapeutic process according to thepresent invention comprises the use of a CD8 peptide, as disclosed inthe present invention, to inhibit specific cells in vivo or in vitro.This process is of particular applicability for purposes of specificimmunosuppression and immunotolerance induction and derives from ournovel finding of CD8's pivotal role in natural immunoregulation. TheCD8-mediated inhibitory effect is contingent upon the simultaneouscopresentation of a molecular signal that normally, in the absence ofCD8, contributes to cellular activation. This second signal can beprovided by a second molecule noncovalently associated with CD8, byvirtue of its presence on the same biomembrane with CD8, or covalentlyassociated with CD8 in an artificial CD8:ligand conjugate. The nature ofthe second, noncovalently or covalently, associated ligand dictates thenature of the target cell to be inhibited. Specific T cells can beinhibited by CD8 in association with allogeneic MHC or an MHC:NAPcomplex. Other specific target cells can be selectively inhibited usingother CD8:ligand combinations as cited (vide supra). The treatment oftarget cells to be inhibited can either be in vitro, prior to infusionof the cell population into the subject, or in vivo, wherein the CD8composition is administered directly to the subject.

As an example of a CD8-mediated immunomodulatory therapeutic process,the sequence of steps that can be executed for inducing tolerance in aprospective transplant recipient for the allogeneic MHC polypeptides ofthe transplant donor, in order to prevent immunological rejection of thegraft following transplantation, are as follows:

(i) Chimeric gene constructs are assembled, within the Bluescriptcloning vector, for producing glycoinositolphospholipid-modified peptidederivatives of human CD8, specific donor allo-class I MHC α heavy chainsand specific donor all-class II MHC α and β chains. In each case, thecoding sequence for the extracellular domain of the respectivepolypeptide is linked in-frame to the 3'-end DAF coding sequence, thelatter encompassing the signals that direct theglycoinositolphospholipid modification process inside of cells. Inaddition, the complete coding sequence for the nonpolymorphic class IMHC β₂ -microglobulin light chain is subcloned into the Bluescriptcloning vector. The PCR-based splice-by-overlap-extension methodology isused, as detailed above, to assemble these genetic constructions, andprimers designed to insert 4 neighboring histidine residues at thepeptide:DAF junction and 3' end of β₂ -microglobulin are employed.

(ii) These coding sequences are nobilized by restriction endonucleasedigestion from the Bluescript cloning vector, using flanking restrictionendonuclease sites in the multiple cloning site of this vector, and eachis inserted into the baculovirus expression vector pVL1392 (obtainedfrom Dr. Max Summers, Texas A&M University) which is suited for genecassettes containing their own translation initiation signals.

(iii) 2 μg of each expression construct is cotransfected into Spodopterafrugiperda (Sf)9 cells in combination with 1 μg Autographa californicanuclear polyhedrosis virus DNA, in order to produce recombinant virusesfor protein expression. By the fourth day post-transfection, up to 50%of the cells have viral occlusions visible in the nucleus and the virustiter is approximately 10⁷ pfu/ml; recombinant viruses account for up to5% of the viral plaques. Purification of viral recombinants is achievedby three rounds of plaque purification.

(iv) Each group of Sf9 cells, infected with plaque-purified recombinantvirus, is harvested, lysed in 1% NP-40 in phosphate-buffered salinecontaining 50 μg/ml of the synthetic elastase inhibitorSuc(OMe)-Ala-Ala-Pro-Val-MCA (Peninsula Laboratories, Inc., Belmont,Calif.) and 1 mM phenylmethylsulfonylfluoride (Sigma Chemical Co., St.Louis, Mo.). Each detergent lysate is passed over a 5 mlnickel-sepharose column, and in each case, a polypeptide mixture, highlyenriched for the respective over-expressed peptide is eluted from thenickel-sepharose matrix by a pH shift, according to the standardprotocol, and dialyzed against neutral buffer. Peptides so produced canbe prepared in advance and packaged into kits.

(v) Unilamellar liposomes coated with glycoinositolphospholipid-modifiedCD8, class Iα, class IIα, class IIβ and unmodified class I β₂-microglobulin are prepared by a detergent dialysis method (see, forexample, Milsmann, M., et al. Biochim. Biophys. Acta 512:147, 1978),wherein a mixture is prepared containing egg lecithin, cholesterol,diacetyl phosphate, and glycoinositolphospholipid-modified peptides in amolecular ratio of 2:1.5:0.2:0.01. The mixture is dissolved in achloroform:methanol solution (2:1) containing 1% sodium cholate, andthis lipid-detergent mixture is subsequently rotary evaporated in around-bottomed flask, depositing a thin dry film. Liposomes formspontaneously when the lipid film is redissolved in phosphate-bufferedsaline (0.1M, pH 7.3). Detergent and excess reagents are removed bydialysis against several changes of 0.05M Tris, pH 7.8, and the finalconcentration of these peptide-coated liposomes is adjusted in Trisbuffer so that phospholipid content is 12 μmol/L. A broad array of U.S.patent filings describe alternative liposomal compositions,incorporating various synthetic lecithins, modified cholesterols andnegative-charged molecules other than diacetyl phosphate, and methodsfor preparing said liposomal compositions, and these can be readilyadapted for preparing CD8-coated liposomes. An alternative to adding theglycoinositolphospholipid-modified peptides to the original mixture ofliposomal constituents is to secondarily incorporate said peptides intoformed liposomes in the presence of 0.003% NP-40. These peptide-coatedliposomes can be stored in a dehydrated state and packaged into kits(see, for example, U.S. Pat. Nos. 4,746,516 and 4,766,046) or usedimmediately for immunomodulation.

(vi) A subject who is to undergo a transplant is assessed foralloreactivity to donor allo-MHC by isolating peripheral bloodmononuclear cells from the prospective transplant recipient's blood, andsetting up a mixed lymphocyte reaction (MLR) with these recipient PBMCas responders and irradiated (5000 R) donor PBMC as stimulators. If asignificant proliferative response is noted, a pharmaceuticalcomposition comprising the peptide-coated liposomes, corresponding todonor allo-MHC, are infused intravenously 6-8 weeks prior to the plannedtransplantation procedure;

(vii) At 3-4 weeks prior to the transplantation date, the in vitro MLRassay is repeated, and if a residual proliferative response betweenrecipient responders and donor stimulators persists, the coated liposomepreparation is reinfused;

(viii) To further enhance engraftment, cells of the graft are coatedwith CD8 prior to transplantation (vide infra).

(ix) The MLR assay is repeated at 6 month intervalspost-transplantation, and booster doses of the CD8 composition areadministered systemically as required.

Of note with respect to this particular example of an immunomodulatoryprocess for prospective transplant recipients are the following: 1)Polymerase chain reaction technology now permits the expeditious cloningof the array of allo-MHCs that are present in the human population, andthis technology further provides for the rapid assembly of MHC.DAF genechimeras. 2) Nominal antigen peptides (NAPs), representing processedpeptides of MHC and other polypeptides, can be added to liposomes orother therapeutic biomembrane preparations bearing CD8 and self-MHC, forimmunomodulation to both allogeneic and other antigens. This approachpermits the treatment of the broad range of autoimmune, allergic andother human diseases where there are unwanted, specific T cellimmunoreactivities. 3) Instead of using liposomes (or other therapeuticbiomembrane preparations) coated with separate CD8 and allo-MHCpeptides, liposomes coated with CD8:MHC (covalent) conjugates or solubleCD8:MHC (covalent) conjugates can be administered to the prospectivetransplant recipient.

The effective subunit valency of the CD8 and ligand components insoluble CD8:ligand conjugates dictates the potency of the biologicaleffect exerted upon target cells. Multivalent conjugates, wherein morethan one CD8 peptide subunit and/or more than one ligand subunit arecovalently linked in each conjugate molecule, are functional equivalentsof membrane-linked multimolecular CD8:ligand combinations. In contrast,univalent conjugates, wherein one CD8 peptide subunit and one ligandsubunit are covalently linked in each conjugate molecule, can, incertain instances, demonstrate lower efficacy. In vitro cellular assays,such as mixed lymphocyte cultures and colony forming assays, that can beused to predict the in vivo effect of a given CD8:ligand conjugate, witha defined subunit valency, are well known to those familiar with theart. Furthermore, in vivo assessment of the activity of a given CD8composition can be performed in a suitable experimental animal. Forinstance, human CD8 compositions can be studied in severe combinedimmunodeficiency disease (SCID) mice reconstituted with human immunesystems.

Another embodiment of a CD8-mediated therapeutic process according tothe present invention comprises the use of CD8:Fc conjugates forgeneralized, nonspecific immunosuppression. These conjugates, in theirsoluble forms, bind to Fc receptors (FcR) on various FcR-bearing cellsin an immunoglobulin isotype-specific fashion. Antigen presenting cells,one set of cells that bear FcRs on their surfaces, can in this way becoated with CD8, and in turn, the antigen-specific activation functionof these cells can thereby be converted to an antigen-specificinhibition function. This, in effect, provides a way to block allantigen presenting cell-dependent immune responses in a general fashion.

Another therapeutic application for CD8:Fc conjugates is the inhibitionof specific FcR-bearing cells. This is of particular relevance for thetherapy of allergic disorders, such as atopic (IgE-mediated) asthma,where Fcε-mediated degranulation of FcεR-positive mast cells andbasophils, leading to the release of mediators such as histamine, is aprimary pathogenetic mechanism. A CD8:Fcε conjugate can be used toeliminate the untoward functional reactivities of these FcεR-positivecells. The Fcε sequence can be derived from either soluble of membrane εheavy chain, and differences in the carboxy termini of these Fcεderivatives can influence regulatory T cell-based molecularinteractions. For example, a CD8-Fcε conjugate, or a therapeuticbiomembrane preparation bearing CD8 and Fcεpeptide units, isadministered parenterally to an allergic subject at 6 month intervals.Allergy testing is performed yearly to monitor the therapeutic response.For subjects with upper airway manifestations of their allergic disease,intranasal and inhalant drug formulations are particularly efficacious.

The use of CD8:Fcε conjugates for inactivating FcεR-bearing cells inallergic subjects represents a specialized application of the moregeneral principle that CD8-mediated inhibition can be applied in apharmaceutical context for inhibiting a broad array of cell types. Forexample, a CD8:monocyte/macrophage-colony stimulating factor (M-CSF)conjugate can be used to inhibit M-CSFR-bearing cells that can normallybe activated by M-CSF. This provides a therapeutic approach for dealingwith clinical conditions in which there is excessive production ofnormal or transformed monocytes. Similarly, CD8:GM-CSF can be used toinhibit granulocyte-macrophage precursors. The nature of the targetcells and the potential clinical applications for the various CD8:ligandconjugates disclosed in the present invention will be apparent to thosefamiliar with the art.

Yet another embodiment of a CD8-mediated therapeutic process accordingto the present invention comprises the use of CD8 peptides to promoteengraftment of cells, tissues and organs, such as kidney, heart, skin,and bone marrow. According to a preferred embodiment of this process,cells of a graft are coated with a membrane-binding CD8 peptide, and thegraft, comprising CD8-coated cells, is then transplanted into therecipient. Glycoinositolphospholipid-modified CD8 peptides aremembrane-binding CD8 compositions well-suited for this purpose, since,peptides so modified spontaneously incorporate into cellular membranesin the presence of low, non-lytic concentrations of detergents (e.g.,0.003% NP-40; J. Exp. Med. 160:1558-1578, 1984). Coexpression of CD8 andallo-MHC on the graft cells serves to inhibit alloreactive T cells, andthereby prolong graft survival through suppression of the rejectionprocess. This process is applicable to a broad array of graft types. Inthe case of a vascularized solid tissue graft, the organ can be perfusedwith a membrane-binding CD8 composition in order to coat the endothelialcells of the graft. Exogenously introduced CD8, in association withendogenously expressed allo-MHC, on graft endothelial cells serves toinhibit allospecific host immune cells entering the tissue or organ andto mitigate acute graft rejection processes directed against theendothelial lining. In the case of bone marrow and other graftscomprised of dispersed cells, CD8-coating of the cells to be engraftedcan be accomplished by combining the cells and a membrane-binding CD8composition together as a suspension in a tissue culture flask. Not onlyare the CD8-coated cells themselves protected from immunologicalrejection, but the transplantation of such CD8-coated graft cells into agiven body compartment further serves to convert said compartment intoan immunologically privileged site for all cells with a sharedallogeneic phenotype. This is of particular utility when long-livedgraft cells are used. For example, allogeneic human bone marrow stromalcells can be coated with CD8 in vitro and transplanted into the host toconvert the host's bone marrow into an immunologically privileged sitefor other cells with a shared allogeneic phenotype that can be engraftedat later times.

The clinical setting of renal transplantation serves to exemplify aCD8-coating process for pretreating a solid organ graft prior totransplantation, in order to block immunological rejection of the graftfollowing transplantation, and the sequence of steps that can beexecuted in this case are:

(i) The kidney of a renal transplant donor is perfused, at the time ofsurgery and prior to its resection, with 1 ml of a solution containing aglycoinositolphospholipid-modified CD8 peptide composition (vide supra)via a bolus injection into the renal artery supplying that kidney, oralternatively, via a bolus infusion into said renal artery by means ofrenal arterial catheterization prior to surgery. The infused solutioncomprises the membrane-binding CD8 peptide in a 0.003% NP-40-containingnormal saline diluent.

(ii) The kidney, once resected, is kept at 4° C. in a perfusatesupplemented with the membrane-binding CD8 peptide in 0.003% NP-40 untiltransplantation into the recipient.

The clinical setting of bone marrow transplantation serves to exemplifya CD8-coating process for pretreating graft cells that are in adispersed state prior to transplantation, in order to blockimmunological rejection of the graft cells following transplantation,and the sequence of steps that can be executed in this case are:

(i) Bone marrow (approximately 15 cc/kg body weight for an adult) isaspirated from a donor by methods well known in the art (see, forexample, U.S. Pat. Nos. 4,481,946 and 4,486,188), and is immediatelyplaced into cold TC-199 medium (Gibco, Inc.) supplemented with heparin(30,000 U/100 ml).

(ii) The bone marrow suspension is centrifuged in a Sorvall RC-3 at 3000rpm, 20', at ambient temperature. The plasma supernatant is separated,and stored for later use. The marrow is transferred to 15 ml sterilepolypropylene tubes, and these are centrifuged at 1500 g, 10', at 4° C.

(iii) The marrow buffy coat is recovered and transferred into a 2000 mltransfer pack for peptide-coating. The nucleated marrow cells areadjusted to a final concentration of 2×10⁷ cells/ml, and hematocrit to<10% with TC-199 medium.

(iv) NP-40 is added to the bone marrow suspension to a finalconcentration of 0.003%, and a glycoinositolphospholipid-modified CD8peptide composition in 0.003% NP-40 is added immediately thereafter. Themixture is incubated for 30' at ambient temperature, and the transferpack is gently agitated every 5'.

(v) The bone marrow cells are transferred into cold satellite bags, andwashed free of detergent and unbound CD8 peptide by serialcentrifugation (RC-3, 2200 g, 4° C., 10' each spin). The marrow cellsare diluted with cold, irradiated autologous plasma to 8×10⁷ nucleatedcells/ml.

(vi) A 2 ml sample is taken for in vitro assays, and processed bydiluting 1:3 with cold TC-199, layering over 3 ml Ficoll-Hypaque(Pharmacia, Inc.), and centrifuging (500 g, 30', ambient temperature).The mononuclear cell layer is thrice washed and assessed for colonyformation capacity in methyl cellulose.

(vii) The remaining CD8-coated cells are mixed with cold freezingsolution [60 ml TC-199+20 ml DMSO (Cryoserv Research IndustriesCorp.)+20 ml irradiated, autologous plasma] at a 1:1 cell ratio. Thecells are then incrementally frozen using computerized cryotechnologicalequipment (e.g., U.S. Pat. Nos. 4,107,937 and 4,117,881) and stored inliquid nitrogen until infusion into the recipient.

The CD8 coating process is applicable to isografts, homografts,heterografts and xenografts. CD8-coated cells, tissues and organsprovide for "universal" donor material, permitting the circumvention ofthe limiting requirement imposed by currently available transplantationtechnologies for histocompatible cells, tissues and organs. For example,CD8-coated epidermal cells can be used in a universal way for skintransplantation. The CD8 coating process can be applied not only tonative, unmodified cells, but also to genetically engineered, orotherwise engineered, cells. This permits the sustained delivery of adefined gene product, to a subject in need of said product, by using aCD8-coated cell as a cellular vehicle that can evade the host'simmunological rejection mechanisms. for example, a transcriptionalcassette, comprising the insulin gene driven by a suitable regulatorypromoter element, can be transfected into human bone marrow stromalcells, and these engineered cells can be coated with CD8 andtransplanted into a diabetic subject, in order to correct such asubject's insulin deficiency. In the particular case of xenografts, theCD8 coating process offers the possibility of generating animal chimerasfor experimental purposes. For example, a mouse can be reconstitutedwith murine CD8-coated human hematopoietic progenitor cells. Thisbypasses the limitation of methodologies currently employed forgenerating mouse-human chimeras, such as the reconstitution of a SCIDmouse with human hematopoietic progenitor cells, which require the useof an immunodeficient, and consequently hard to maintain, mouse as ahost for the human cells.

As described above, in addition to coating the graft cells with CD8,engraftment can be enhanced by pretreating the graft recipient withtherapeutic biomembrane preparations bearing CD8 and allo-MHC or withsoluble CD8:MHC conjugates prior to transplantation, in order to inducespecific immunotolerance to the allo-MHC of the transplanted cells,tissues or organs. Biomembrane compositions that can be used for thispurpose comprise native or engineered cells, liposomes, planarmembranes, or pseudocytes. CD8:MHC conjugates comprising both class Iand class II MHC components can be coadministered.

Still another embodiment of a CD8-mediated therapeutic process accordingto the present invention comprises the use of CD8 peptides to preventgraft versus-host disease when bone marrow is transplanted to anon-identical recipient. A therapeutic biomembrane preparation, bearingboth CD8 and host MHC peptides or a CD8:MHC conjugate is added to donorbone marrow cells in vitro, and following variable incubation periods,the cells are infused into the transplant recipient. This form oftreatment eliminates alloreactive immune cells amidst the donor bonemarrow cells, and mitigates the requirement for T-cell depleting thebone marrow.

In the clinical situation of bone marrow transplantation, the variousCD8-dependent inhibitory processes described heretofore can be combinedin a multifacted way to inhibit both host-versus-graft andgraft-versus-host responses. As an example, a sequence of steps that canbe executed in this setting are:

(i) A CD8 peptide composition, comprising CD8 and donor allo-MHC, isadministered to the transplant recipient 6-8 weeks prior totransplantation, with a booster dose at 3-4 weeks, if required, in orderto suppress alloreactive cells of the recipient.

(ii) Bone marrow is aspirated from the donor, NP-40 is added to thesuspension to a final concentration of 0.003%, and a CD8 peptidecomposition, comprising CD8 and recipient allo-MHC, is added to the bonemarrow cells, incubated for 4 h at 37° C., in order to inhibitalloreactive cells of bone marrow cell population.

(iii) Bone marrow cells are coated with a CD8 peptide composition,comprising a membrane-binding CD8 peptide, and the cells are eitherstored in cryopreservative in liquid nitrogen until use or immediatelyinfused into the transplant recipient.

The compositions active in the novel methods of treatment of thisinvention can be administered in a wide variety of therapeutic dosageforms in conventional vehicles. A variety of effective formulations forpeptide pharmaceuticals, as well as dosing schedules forimmunomodulatory agents, are known to those familiar with the art andcan be applied to the CD8 peptides disclosed heretofore. Non-immunogeniccarriers, such as carboxymethyl cellulose (U.S. Pat. No. 4,415,552), areparticularly well suited for soluble CD8 compositions. Therapeuticcellular preparations for CD8-based therapy are infused intravenouslyinto the subject. Therapeutic liposome and other planar membranepreparations for CD8-based therapy can be administered parenterally ororally. Modulation of target cells is accomplished by administering to asubject, or treating cells of a subject in vitro, with a dose, or seriesof doses which will achieve the desired modulatory effect. The efficacyof cellular modulation, such as the degree of CD8-mediatedimmunosuppression, can be easily monitored using conventional in vivoand in vitro immunological testing methods, and booster doses can beadministered as needed.

It is understood that various other modifications will be apparent toand can readily be made by those skilled in the art without departingfrom the scope and spirit of this invention. Accordingly, it is notintended that the scope of the claims appended hereto be limited to thedescription as set forth above, but rather that the claims be construedas encompassing all the features of patentable novelty, ensuing from thedisclosure of CD8's inhibitory ligand activity, which would be treatedas equivalents thereof by those skilled in the art to which thisinvention pertains.

We claim:
 1. A method for specifically reducing T cell proliferation orcytotoxicity, directed to alloantigens or processed antigens, comprisingthe steps of:(a) providing a non-naturally occurring antigen presentingcell which presents in, or on its surface the extracellular domain ofCD8 and said alloantigens or processed antigens, and (b) exposing said Tcells capable of responding to said alloantigens or processed antigens,for a time, and under conditions sufficient to reduce the specificcellular immune response of said cell T cells to said alloantigens orprocessed antigens.
 2. The method of claim 1 wherein said non-naturallyoccurring antigen presenting cell is contacted in vivo with said Tcells.
 3. The method of claim 1 wherein the non-naturally occurringantigen presenting cell comprises a chimera consisting essentially ofthe extracellular domain of CD8 covalently-linked to an immunoglobulinFc domain.
 4. The method of claim 1 wherein the non-naturally occurringantigen presenting cell comprises a chimera consisting essentially ofthe extracellular domain of CD8 covalently-linked to aglycoinositolphospholipid moiety.
 5. A method of making a non-naturallyoccurring antigen presenting cell, which expresses the extracellulardomain of CD8 on its surface, capable of specifically reducing T cellproliferation or cytotoxicity, directed to alloantigens or processedantigens, comprising the steps of:(a) isolating a eukaryotic cellcomprising alloantigens to which a reduced cellular response is desired,and (b) transforming said cell with a gene encodingglycoinositolphospholipid-modified CD8, wherein said gene expressesglycoinositolphospholipid-modified CD8 in a manner which presents theextracellular domain of CD8 on the surface of said cell.
 6. A method ofmaking a non-naturally occurring antigen presenting cell, capable ofspecifically reducing T cell proliferation or cytotoxicity, directed toalloantigens or processed antigens, comprising the steps of:(a)isolating a cell comprising alloantigens or processed antigens, and (b)contacting said cell with a chimera for a time, and under conditionssufficient for binding of said chimera to the surface of said cell,wherein the chimera consists essentially of the extracellular domain ofCD8 and a moiety not naturally associated with CD8, said moietysufficient to bind said chimera to the surface of said cell in a mannerwhich presents the extracellular domain of CD8 on said cell's surface.7. A method of making a non-naturally occurring antigen presenting cell,capable of specifically reducing T cell proliferation or cytotoxicity,directed to alloantigens or processed antigens, comprising the stepsof:(a) isolating a cell comprising alloantigens or processed antigens,and Fc receptors, and (b) contacting said cell with a chimera,consisting essentially of the extracellular domain of CD8 and animmunoglobulin Fc domain, for a time and under conditions sufficient forbinding of said conjugate to the surface Fc receptors of said cell.